Reaction products of propane-1,2-diamine with 2-[(2-methylphenoxy)methyl]oxirane

Endpoint summary

Description of key information

Based on the LLNA data available, the test item was considered a moderate skin sensitizer (EC3 > 2 %).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-11 to 2016-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90. Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9-11 weeks old (at start of the main test)
- Weight at study initiation: 17.3 – 20.6 g
- Housing: Grouped caging (5 animals/cage)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

propylene glycol

Concentration:

10%, 5%, 2.5% and 1% (w/v)

No. of animals per dose:

5 females per group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item is a viscous liquid, which was adequately miscible with the vehicle (propylene glycol).
- Irritation: Ear thickness was greater or equal than 25% at high test concentrations (75%, 50% or 25%, w/v) in the first range finding test. No irritation was observed at 10%, 5%, 2.5% and 1% (w/v) test item formulations in the second range finding test.
- Lymph node proliferation response: No assessement of lymph node proliferation was done.
- Other: Mortality (1/2 animals) was observed in the 75% (w/v) dose group on day 5. Systemic toxicity (decreased body weight, loss of hair, scab) was observed at high concentrations (75 %, 50 % or 25 %, w/v) in the first range finding test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- In the main test 35 female CBA/Ca Ola Hsd mice were allocated to 7 groups of 5 animals each:
- 4 groups received the appropriate formulation of the test item at concentrations of 10%, 5%, 2.5% and 1% (w/v)
- two negative control groups were used: 1. vehicle control for the test item (propylene glycol) and 2. vehicle control for positive control (acetone:olive oil 4:1 (AOO))
- the positive control group received hexyl cinnamic aldehyde (25% (w/v) in AOO)
- Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, of the positive control substance and of the vehicle. The formulations were applied, with a pipette, on the dorsal surface of each ear.
- Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There were no treatments on Days 4, 5 and 6.

- Criteria used to consider a positive response: That exposure to at least one concentration (non-irritating, non-toxic) of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION:
The test item was weighed and formulations prepared with the vehicle in a final volume of 1 mL using volumetric vials and mechanical agitation. All formulations were freshly prepared just before the daily treatments.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The measured DPM values corrected with the mean background value were used for statistical analysis of the proliferation data. The heterogeneity of variance between the groups treated with the test item and the vehicle control (propylene glycol) was checked by Bartlett's test. Since significant heterogeneity was detected, the normal distribution of data was examined by Kolmogorow-Smirnow test followed by the non-parametric method of Kruskal-Wallis One-Way analysis of variance. As a results of this analysis the inter-group comparisons was performed using Mann-Whitney U-test to assess the significance of inter-group differences. Significance of the positive control response was evaluated by t-test versus the relevant vehicle control (acetone:olive oil 4:1 mixture). Significance of the dose-response was evaluated by linear regression made with Microsoft Excel Software.

Positive control results:

The positive control substance induced the appropriate stimulation (an SI ≥ 3) compared to the relevant (AOO) control: the calculated SI value was 9.0.

Key result

Parameter:

SI

Value:

26.3

Test group / Remarks:

Dose group 10 % in PG

Key result

Parameter:

SI

Value:

8.9

Test group / Remarks:

Dose group 5 % in PG

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

Dose group 2.5 % in PG

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Dose group 1 % in PG

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control for test item: PG

Key result

Parameter:

SI

Value:

9

Test group / Remarks:

Positive control: 25 % HCA in AOO

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control for the positive control: AOO

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Visually larger lymph nodes compared to the relevant vehicle controls (AOO or PG) were observed in the positive control group and in the 10 % or 5 % (w/v) test item treated groups. Visual appearance of the lymph nodes was normal in the negative (vehicle) control groups and in the 2.5 % or 1 % (w/v) test item treated groups. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at test concentrations of 10 % and 5 % (w/v). No significant lymphoproliferation was observed at the other test concentrations (2.5 % or 1 %, w/v). The corresponding stimulation index values were 26.3, 8.9, 1.1 and 1.0 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively.

CLINICAL OBSERVATIONS
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or other local effects were observed in any treatment groups.

BODY WEIGHTS
No significant, treatment related effect on the body weights was observed in any treatment group.

1. Range finding test

Test item residuals were observed on the ears in all dose groups which made surface of the ears sticky. It is considered that the measured ear thickness values (especially on day 5) could be affected by the test item residuals also.

Table 1: Mean DPM and DPN values for all groups in the main test

Test group	Mean DPM (SD)	Mean DPN (SD)
Vehicle control (AOO)	1229.7 (92.6)	615 (46.3)
Positive control (HCA)	11091.9 (6837.4) * T	5546 (3418.7)
10% test item in PG	22222.3 (10511.2) ** U	11111 (5255.6)
5% test item in PG	7509.7 (5694.1) ** U	3755 (2847.1)
2.5% test item in PG	888.5 (542.0) NS U	444 (271.0)
1% test item in PG	824.5 (578.9) NS U	412 (289.5)
Vehicle control (PG)	843.9 (567.6)	422 (283.8)

HCA = α-Hexylcinnamaldehyde

AOO = Acetone: Olive oil 4:1 (v/v) mixture

PG = Propylene glycol

* = Significant; p < 0.05

** = Significant; p < 0.01

NS = Not significant

T = t-test versus control (AOO)

U = Mann-Whitney U-test versus control (PG)

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Based on the EC3 value of 3.1%, the test item was considered a moderate skin sensitizer in this LLNA(EC3 > 2 %).

Executive summary:

The aim of this study (OECD 429) was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Individual approach was used in this test. The test item was a viscous liquid. Propylene glycol (PG), one of the vehicles preferred by the relevant guidelines, was selected to be used for formulation. The test item was adequately miscible with the vehicle. The maximum dose selection based on results of two consecutive Dose Range Finding (DRF) tests performed according to the relevant guidelines. Adverse effects (systemic toxicity and irritation) was observed at high test concentrations (75 %, 50 % or 25 %, w/v), hence the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as formulations in PG. Appropriate positive control, furthermore two negative control groups (dosed with the vehicles of the test and positive control groups, respectively) were employed. The positive control item (α-Hexylcinnamaldehyde [HCA]; 25 % (w/v) in AOO) induced the appropriate stimulation over the control (SI = 9.0), thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or other sign of systemic toxicity were observed in any treatment group. No visible signs of irritation or other local effect were observed in any treatment groups. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at test concentrations of 10 % and 5 % (w/v). No significant lymphoproliferation was observed at the other test concentrations (2.5 % or 1 %, w/v). The corresponding stimulation index values were 26.3, 8.9, 1.1 and 1.0 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. The measured individual DPM values corrected with the mean background value were statistically evaluated. Statistically significant increase of the proliferation values was observed in the positive control group by t-test versus AOO control (p < 0.05). Statistically significant differences compared to the vehicle control (PG) were observed in the 10% and 5% (w/v) test groups (p < 0.01, evaluated by Mann-Whitney U-test). The dose-response relationship, evaluated by linear regression using the SI values, was statistically significant (p = 0.015, r = 0.98). According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation observed at 10 % and 5 % (w/v) concentrations and the significant dose-response correlation are considered evidence that the test item is a skin sensitizer.

The calculated EC3 value based on dose-response curve analysis was 3.1 % (w/v) in this LLNA. Based on the EC3 value of 3.1%, the test item was considered a moderate skin sensitizer in this LLNA(1 ≤ EC3 ≤ 10).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The aim of this study (OECD 429) was to determine the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Individual approach was used in this test. The test item was a viscous liquid. Propylene glycol (PG), one of the vehicles preferred by the relevant guidelines, was selected to be used for formulation. The test item was adequately miscible with the vehicle. The maximum dose selection based on results of two consecutive Dose Range Finding (DRF) tests performed according to the relevant guidelines. Adverse effects (systemic toxicity and irritation) was observed at high test concentrations (75 %, 50 % or 25 %, w/v), hence the test item was examined in the main test at 10 %, 5 %, 2.5 % and 1 % (w/v) concentrations as formulations

in PG. Appropriate positive control, furthermore two negative control groups (dosed with the vehicles of the test and positive control groups, respectively) were employed. The positive control item (α-Hexylcinnamaldehyde [HCA]; 25 % (w/v) in AOO) induced the appropriate stimulation over the control (SI = 9.0), thus confirming the validity of the assay. No mortality was observed during the main test. No significant, treatment related effect on body weights or other sign of systemic toxicity were observed in any treatment group. No visible signs of irritation or other local effect were observed in any treatment groups. Significant lymphoproliferation (SI ≥ 3) was observed for the test item at test concentrations of 10 % and 5 % (w/v). No significant lymphoproliferation was observed at the other test concentrations (2.5 % or 1 %, w/v). The corresponding stimulation index values were 26.3, 8.9, 1.1 and 1.0 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. The measured individual DPM values corrected with the mean background value were statistically evaluated. Statistically significant increase of the proliferation values was observed in the positive control group by t-test versus AOO control (p < 0.05). Statistically significant differences compared to the vehicle control (PG) were observed in the 10% and 5% (w/v) test groups (p < 0.01, evaluated by Mann-Whitney U-test). The dose-response relationship, evaluated by linear regression using the SI values, was statistically significant (p = 0.015, r = 0.98). According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation observed at 10 % and 5 % (w/v) concentrations and the significant dose-response correlation are considered evidence that the test item is a skin sensitizer. The calculated EC3 value based on dose-response curve analysis was 3.1 % (w/v) in this LLNA. Based on the EC3 value of 3.1%, the test item was considered a moderate skin sensitizer in this LLNA (1 ≤ EC3 ≤ 10).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on sensitisation properties, the test item is classified and labelled as a skin sensitiser cat. 1 B (H317: May cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.